Interpretation of immunohistochemistry (IHC) data is difficult in the absence of appropriate controls.
To confirm staining specificity and instill confidence in your results, a positive and negative tissue control should be routinely included in IHC experiments.
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Need help identifying a positive and negative control for your IHC...
Tyrosine hydroxylase is a member of the aromatic amino acid hydroxylase (AAAH) family. It is expressed throughout the central nervous system (CNS) and catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which can be, through a series of downstream enzymatic reactions, processed into the neurotransmitter and signaling molecule dopamine. Dopamine can then be further altered to produce norepinephrine or epinephrine. Tyrosine hydroxylase is the rate limiting enzyme in this pathway, also referred to as the catecholamine synthesis pathway.
Antibodies that detect tyrosine hydroxylase are often used to identify dopaminergic neurons in the CNS. In the mammalian retina, for instance, a subset of dopaminergic amacrine cells that form a single synaptic strata in the inner retina specifically express tyrosine hydroxylase and are often identified through tyrosine hydroxylase antibody staining (Wulle and...
Actin is an abundant cytoskeletal protein involved in a variety of cellular processes such as cell motility, cell division, and muscle contraction. Actin monomers assemble into filaments and can provide a track for transport of cargo by the molecular motor myosin (1). Alternatively, interaction with myosin allows contraction between actin filaments. This contractility is essential during cell migration and cytokinesis (1). On a larger scale, organized scaffolds of actin filaments interact with myosin to provide mechanical force during muscle contraction (1).
Actin antibodies are widely used as loading controls when measuring protein levels by performing western blots (3). While actin often serves as a normalization control in westerns or qPCR, actin antibodies also serve as important research tools for assessing the cytoskeletal network of cells or muscle fibers. For these purposes...
Microtubules are a main component of the cytoskeleton and play essential roles in a variety of cellular processes. These highly dynamic tubular structures are assembled from alpha- and beta-tubulin dimers to form a complex structural network of microtubules throughout the cytoplasm. This network provides a substrate for intracellular trafficking of vesicles, organelles, and other cargo and can also facilitate cell migration. Additionally, during cell division, microtubules make up the mitotic spindle and provide the mechanical force for chromosome segregation. The diversity of microtubule functions is made possible by the large number of binding partners as well as regulation through post-translational modification. Microtubule associated proteins (MAPs), such as kinesin or dynein, can bind to tubulin and transport cargo while others alter microtubule stability. The tubulin family of proteins consists of many isoforms, each with specialized roles within the cell....
Autophagy is a cellular mechanism used to regulate cell metabolism and to recycle or degrade damaged organelles and proteins. This is accomplished through the engulfment of cargo in a double-membrane structure called the autophagosome. The autophagosome fuses with the vacuole or the lysosome where hydrolytic enzymes facilitate the degradation of biomolecules. Each step of autophagy is a tightly regulated process from the recognition of cargo to the assembly of the autophagosome. ATG11 is an important scaffolding protein that seems to be involved in selective autophagy. Specifically, ATG11 facilitates the fusion of autophagosomal vesicles and the vacuole by linking selective autophagy receptors with a multiprotein complex consisting of ATG1-ATG13 (1). The ATG1-ATG13 complex is inactivated under normal conditions. Starvation triggers the binding of ATG17-ATG31-ATG29 to assemble a large pentameric complex capable of facilitating autophagosome...
Conjugated antibodies are essential research tools for countless cell and molecular assays. While much of the time a researcher’s needs can be met with the wide variety of commercially available antibody conjugates, some applications may call for a unique reagent. For this reason some scientists opt for the flexibility of conjugating their own antibodies. While this approach may seem daunting, the availability of antibody labeling kits like the Novus Lightning-Link Kit have simplified the conjugation process and reduced the time needed to generate your own high quality antibody reagents. The Lightning-Link reagent kit allows you to efficiently label your antibody in just 30 seconds. Whether you are simply trying to save your lab a bit of money or synthesizing a unique reagent with no commercial alternatives, take into account the...
Beta-Actin is a highly conserved protein involved in cell growth, cytoskeletal and extracellular support structures and cell migration. Because beta-Actin is ubiquitously expressed in all eukaryotic cells, it is frequently used as a loading control for assays involving protein detection, such as Western blots.
Interestingly, Beta-Actin has been shown to be associated with growth cones in developing neurite cells. While beta-Actin is one of several actins involved in the guidance of growth cones towards synaptic targets, beta-Actin mRNA has also been shown to regulate protein synthesis by binding to Vg1RBP, ultimately resulting in asymmetrical translation of beta-Actin near the site of growth cone turning.1 Beta-actin has also been shown to localize in areas associated with neuronal growth and remodeling in both developing and mature neuronal cell types. Beta-actin is primarily found in the growth cones, filopodia, cell bodies and...
Diabetes is a disease that impacts over 29 million people in the US (1). It is metabolic disorder that is the result of a high level or sugar or glucose in the blood. The three types of diabetes are Type 1, Type 2 and gestational diabetes, with Type 2 being the most common worldwide (2). The factors that contribute to a person getting Type 1 or Type 2 are largely unknown, but genetics, obesity and exercise have been linked to diabetes (3). Learn more about diabetes in our infographic below.
Novus Biologicals offers 3,700+ reagents for the research of diabetes.
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ChREBP (carbohydrate response element-binding protein) is a glucose responsive basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that binds MLX and then carbohydrate response element /ChoRE for the induction of genes involved in glycolysis, de novo lipogenesis (DNL), and fatty acid desaturation. ChREBP’s target genes includes glucokinase (GCK), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), pyruvate kinase/liver type pyruvate kinase (PK1/ PKLR), delta-9-desaturase (SCD/SCD1) etc. ChREBP is expressed in a number of mammalian tissues such as liver, skeletal muscle, white/brown adipose, heart, kidney, cerebellum and intestine. Increased hepatic ChREBP expression has been suggested to result in development of non-alcoholic fatty liver disease (NAFLD) and obesity through the conversion of carbohydrates into triglycerides. Recent findings have suggested that besides glucose-lipids homeostasis, it implicates in pathways linked to...
Copper is an essential micronutrient that serves as a cofactor in numerous biological processes, but can be toxic when present in excess. Because of this, cells must tightly maintain copper levels. This includes balance between import and export of cellular copper. The major copper importer in humans is the high-affinity copper transporter SLC31A1 or CTR1. The localization of CTR1 varies between cell types, but is commonly found at the plasma membrane and intracellular vesicles [1-3].
In addition to copper, CTR1 has the ability to transport platinum-containing drugs. One example is a chemotherapeutic drug called cisplatin that is used to treat a variety of cancers. Unfortunately, many types of cancer are resistant to platinum drugs, presenting a major obstacle for achieving maximum drug efficacy. In yeast, western blotting using a CTR1...
Trex1 the protein is involved in DNA damage response. Tyrannosaurus rex (T. rex) the dinosaur lived during the Cretaceous Period. Raptor the protein is a regulator of mTOR activity. Velociraptors the dinosaurs lived during the Cretaceous Period. Learn more about Trex1 and Raptor proteins as well as some fun facts about the tyrannosaurus rex and the velociraptor in our infographic below.
Novus Biologicals offers reagents for these proteins for your research needs:
EEA1, or early endosome antigen 1, is a membrane bound Rab5 effector protein specific to the early endosome and plays an important role in membrane trafficking. Early endosomes fuse with endocytic vesicles to redistribute compounds to other cellular destinations. EEA1 contains a C-terminal FYVE domain that binds to phosphatidylinositol-3-phosphate, which targets it to early endosomes [1, 2]. EEA1 is essential for early endosome docking together with SNARES contained on endocytic vesicles, allowing for membrane fusion [3]. ...
Fluorescent probes conjugated to antibodies allow for simultaneous IHC detection of multiple antigens in the same tissue section. However, quite often conventional multi-color IHC cannot be done if only primary antibodies raised in the same host species are available to the researcher. To solve this problem, we developed a novel technique for performing multicolor fluorescence immunohistochemistry using primary antibodies derived from a single host source. Alex Kalyuzhny, PhD, Immunohistochemistry and ELISpot Manager at Bio-Techne, will discuss modifications of primary antibodies allowing using fluorescent secondary antibodies so they bind only to primary antibodies of interest but don’t cross-react with irrelevant primary antibodies.
Register now for our FREE webinar on Thursday, January 14, 2016.
The c-Jun N-terminal kinase (JNK) family is a group of regulatory kinases with important functions in cell morphogenesis, inflammation, differentiation, and cell death (1). Aberrant activation of JNK family proteins in cancers has led to interest in small molecule JNK inhibitors as a therapeutic strategy (1). JNK1, also known as MAPK8, is expressed in most tissues and is involved in transduction of extracellular signals such as growth factors or cytokines though a phosphorylation cascade to elicit diverse intracellular responses (1). JNK phosphorylation substrates include p53, AP-1, c-Myc, and Bcl-2 (1). Defects in JNK signaling have been observed in inflammatory and neurodegenerative disorders (1). For example, increased JNK1 activity leads to hyperphosphorylation of tau in Alzheimer’s disease (2). A recent study reduced JNK1 activity by using heterozygous JNK1 deficient mice and...
The inflammatory response consists of a complex network of signaling pathways that regulate a diverse set of cytokines, growth factors, adhesion molecules, and transcription factors (1). Of the proinflammatory signaling pathways the NF-kB family is particularly well studied for its role in apoptosis, cancer, and the development and maintenance of the immune system (1). The family consists of the transcription factors p50, p52, RelA (p65), RelB, and c-Rel. Each of these share the Rel homology domain (RHD) that is responsible for dimerization and DNA binding (2). Under normal conditions NF-kB exists as a cytoplasmic dimer in an inactive state associated with the IkB proteins (2). Stimuli, such as a microbial infection, activate TNF receptor or Toll-like receptors to activate the IkB kinase complex leading to the ubiquitination...
Mitogen-activated protein kinases (MAPKs) are important signaling proteins needed to transmit and relay extracellular stimuli and to illicit intracellular responses (1). The MAPK family of proteins are serine/threonine kinases that are able to phosphorylate and activate downstream kinases in a signal cascade that regulates diverse cell responses such as gene expression, metabolism, apoptosis, and differentiation (1). Notable members of the MAPK family include ERK, JNK, and p38 (1). Misregulation of these pathways has been implicated in neurodegenerative diseases as well tumorigenesis (1). ERK1, also known as MAPK3, is one of the downstream kinases activated by receptor-tyrosine kinases such as EGFR and the Ras GTPase (2). ERK1/2 can phosphorylate hundreds of substrate proteins including transcription factors and cytoplasmic regulators (2). Additionally, increased...
Histones make up the main protein component of chromatin and are responsible for storing and organizing the genome in a compact yet accessible manner. In addition to storage, histones play an important role in the regulation of various cellular processes such as DNA replication, transcription, and mitosis by regulating the accessibility of DNA to various DNA-binding proteins. Simply put, chromatin exists in “open” and “closed” states. Chromatin in its closed state is inaccessible to DNA-binding proteins like transcription factors while open chromatin is accessible to DNA replication and RNA transcription machinery. Transitions between open and closed chromatin states are carried out through the eviction of nucleosomes or by altering DNA-histone interactions through the post-translational modification of histone proteins. These reactions are carried out by chromatin remodeling complexes. Histone H4 is one of the core structural histones that...
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a well-known housekeeping gene with functions in glycolysis. Many biologists are familiar with the gene and use GAPDH antibodies for a loading control when performing western blots. However, this primarily cytoplasmic protein is an essential metabolic regulator and has been shown to be involved in a variety of cellular processes like DNA repair, membrane fusion, and cell death (1). Cytoplasmic GAPDH exists as a tetramer and normally mediates the formation of ATP and NADH during glycolysis (1). Under oxidative stress GAPDH can be post-translationally modified to regulate cell metabolism (1). Additionally GAPDH has been shown to interact with the cytoskeleton to influence microtubule and actin polymerization (1).
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Histones are nuclear proteins essential for the storage and organization of genomic DNA as chromatin. Chromatin consists of DNA wrapped tightly around histone oligomers to form nucleosomes. In addition to compacting the genome, histones also regulate the accessibility of the DNA to the transcription and replication machinery to influence gene expression and mitosis. In addition to the core histones H1, H2A, H2B, H3, and H4, there are histone variants with specific functions in gene expression, development, and DNA repair. One of the variants is HIST3H3, also known as H3.1t or H3t. The H3.1t protein is primarily expressed in the testis leading researchers to believe its main function is in DNA storage and compaction during spermatogenesis (1). Analysis of H3.1t containing nucleosomes revealed inherent instability compared to...
Myc is a basic helix-loop-helix zipper transcription factor that regulates a network of many hundreds of genes. Myc up-regulates the expression of many genes involved in cell growth and proliferation such as ribosome biogenesis and protein synthesis (1). While many Myc induced genes are transcribed by RNA polymerase II, tRNA and rRNA genes are also Myc targets (1). Myc is also responsible for repressing genes involved in cell-cycle arrest and cell adhesion. Genome-wide profiling of Myc binding through chromatin immunoprecipitation with Myc antibody has revealed broad association with many targets with some estimates as high as 10-15% of genomic loci (2). The ability of Myc to activate or repress such a large number of targets allows integration of environmental signals and regulation of opposing cellular pathways (1). The ability to enhance cell growth and block cell-cycle arrest makes Myc an important proto-oncogene as well. The...
Histones are the main protein component of chromatin and are essential for the storage and compaction of the genome. DNA wraps around histone oligomers to make up nucleosomes, the individual subunits of chromatin. By altering the accessibility of the genome, chromatin structure is important for regulating various cellular processes including replication, transcription, and DNA repair. Typically chromatin structure is influenced by post-translational modification of histone proteins at lysine and arginine residues. These residues are concentrated at the amino-terminal end of the histone protein and can alter its interaction with the DNA or recruit and bind to chromatin remodeling complexes. The complex language of histone modifications creates multiple levels of gene regulation and also forms the basis for epigenetic regulation. Of the core histones, H2A, H2B, H3, and...
Histones, the main protein component of chromatin, are essential for storing and organizing the genome in a compact yet accessible manner. DNA wraps tightly around histone oligomers to form nucleosomes which can store unused portions of DNA and regulate accessibility to the replication or transcription machinery. By affecting chromatin structure histones and their modifying enzymes make up a complex network with important regulatory roles in DNA replication, mitosis, and gene expression. In addition to the canonical histones 1-4, there are specialized histone variants with important regulatory functions in transcription or DNA repair for example. The histone variant H3.3 is deposited during replication independent assembly of nucleosomes and is typically enriched in active genes. Although only differing from canonical H3 by four amino acid residues, H3.3 can play important functions in gene expression and differentiation (1). These...
Thyroid transcription factor 1 (TTF-1), also known as NKX2.1, is a conserved master regulatory transcription factor involved in the development of the lung, brain, and thyroid (1). In the lung TTF-1 positively regulates the expression of several lung-specific proteins including thyroglobulin, thyroperoxidase, and surfactant proteins A, B, and C (1). TTF-1’s control of this transcriptional network is important for the terminal differentiation of various lung cell lineages (1). Detection of the nuclear TTF-1 through immunostaining with TTF-1 antibodies has served an important role in dissecting the protein’s function during the morphogenesis and differentiation of epithelial lung cells (2). In addition to its normal function in differentiation, TTF-1 is also...
The inflammasome is a multi protein complex that is an important component of the innate immune response. The inflammasome is able to sense and respond to pathogen infections by recognizing pathogen-associated molecular patterns and mediating the secretion of inflammatory cytokines. Of the various types of inflammasomes, NLRP3/NALP3 is unique because of the diverse range of microbes it is able to detect (1). Once assembled and activated, the NLRP3/NALP3 inflammasome mediates the caspase-1 dependent activation of into its active secreted form interleukin-1 into its active secreted form (1). Although important for responding to pathogen infections, the NLRP3/NALP3 inflammasome is also involved in chronic inflammatory and age-related diseases such as atherosclerosis, Alzheimer's, and inflammatory bowel disease (1). Misactivation of the NLRP3/NALP3 inflammasome contributes to these diseases and may be...
AKT1 is a serine/threonine protein kinase with homology to protein kinase A (PKA) and protein kinase C (PKC). AKT1 contains the central kinase domain sandwiched between a pleckstrin homology domain and a regulatory domain (1). AKT1 is regulated by receptor tyrosine kinase pathways and is activated in a PI3K-dependent manner following growth factor stimulation (1). Production of PIP3 at the plasma membrane by PI3K is though to recruit AKT1 where it is then phosphorylated by 3-phosphoinositide-dependent kinase 1 (PDK1). Activated AKT1 mediates a variety of cellular processes by phosphorylating downstream substrates to affect their activity, localization, or stability (1). These cellular processes include regulation of cell metabolism, growth, survival, and cell cycle progression (1). Mutation of AKT1 and deregulation of these cellular processes can lead to various diseases such as...
